U.S. patent application number 15/174633 was filed with the patent office on 2016-12-08 for multiplexed spectral lifetime detection of phosphors.
The applicant listed for this patent is INTELLIGENT MATERIAL SOLUTIONS, INC., LEIDEN UNIVERSITY MEDICAL CENTER. Invention is credited to Howard Y. BELL, Joshua E. COLLINS, Paul L.A.M. CORSTJENS, Sukwan HANDALI, Hans J. TANKE.
Application Number | 20160356780 15/174633 |
Document ID | / |
Family ID | 57452326 |
Filed Date | 2016-12-08 |
United States Patent
Application |
20160356780 |
Kind Code |
A1 |
BELL; Howard Y. ; et
al. |
December 8, 2016 |
MULTIPLEXED SPECTRAL LIFETIME DETECTION OF PHOSPHORS
Abstract
New methods and assays for multiplexed detection of analytes
using phosphors that are uniform in morphology, size, and
composition based on their unique optical lifetime signatures are
described herein. The described assays and methods can be used for
imaging or detection of multiple unique chemical or biological
markers simultaneously in a single assay readout.
Inventors: |
BELL; Howard Y.; (Princeton,
NJ) ; COLLINS; Joshua E.; (Philadelphia, PA) ;
CORSTJENS; Paul L.A.M.; (Leiderdorp, NL) ; HANDALI;
Sukwan; (Norcross, GA) ; TANKE; Hans J.;
(Rijnsburg, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INTELLIGENT MATERIAL SOLUTIONS, INC.
LEIDEN UNIVERSITY MEDICAL CENTER |
Princeton
Leiden |
NJ |
US
NL |
|
|
Family ID: |
57452326 |
Appl. No.: |
15/174633 |
Filed: |
June 6, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62171320 |
Jun 5, 2015 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/582 20130101;
G01N 2458/40 20130101 |
International
Class: |
G01N 33/58 20060101
G01N033/58 |
Claims
1. A method for detecting one or more analytes in a sample,
comprising the steps of: (a) contacting the sample with two or more
types of phosphor particles, wherein each type of phosphor is
conjugated to a capture molecule specific for an analyte of
interest, to separately bind and label each analyte of interest;
(b) separating phosphor particles bound to the analytes from
unbound phosphor particles; and (c) detecting each labelled analyte
by the unique optical lifetime signature of the corresponding
phosphor; wherein the phosphor particles conjugated to each type of
capture molecule have unique and uniform morphology, size, and/or
composition.
2. The method of claim 1, wherein the detecting step is performed
in a single readout.
3. The method of claim 1, further comprising, before step (a), the
step of capturing the one or more analytes on an analyte-specific
capture molecule attached to a substrate.
4. The method of claim 1, wherein the phosphor particles are
up-converting phosphor particles comprising at least one rare earth
element and a phosphor host material.
5. The method of claim 1, wherein the method is cell sorting method
and the analyte of interest is a cell.
6. The method of claim 1, wherein the method is used in flow
cytometry.
7. The method of claim 1, wherein the phosphor particles are about
30 nm to about 400 nm in size.
8. The method of claim 1, wherein the sample comprises a bodily
fluid.
9. The method of claim 8, wherein the sample comprises blood serum,
saliva, tissue fluid, or urine.
10. An assay kit for detecting one or more analytes in a sample,
comprising two or more types of phosphor particles conjugated to
capture molecules specific for each analyte, wherein the phosphor
particles conjugated to each type of capture molecule have unique
and uniform morphology, size, and/or composition.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Patent Application
62/171,320, filed Jun. 5, 2015, the disclosure of which is
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to methods and assays for multiplexed
detection and serialization of analytes using detectable phosphor
labels where particular monodisperse phosphor particles are
characterized by a uniform morphology, a uniform size, and/or
composition and possesses their own unique optical spectral
lifetime signatures.
BACKGROUND OF THE INVENTION
[0003] The invention relates generally to detectable labels and
compositions useful in assay methods for detecting soluble,
suspended, or particulate substances or analytes such as proteins,
carbohydrates, nucleic acids, bacteria, viruses, and eukaryotic
cells and more specifically relates to compositions and methods
that include luminescent (e.g., phosphorescent) labels.
[0004] A detectable phosphor label is typically a phosphor
conjugated with capture molecules that are specific for analytes of
interest. Detectable phosphor labels can be used in all assay
applications where fluorochrome, enzyme, or isotope-labelled
immuno-reagents are used. Various phosphor conjugates, their
preparation, and use were previously described in, for example,
U.S. Pat. No. 5,043,265 (Tanke et al.), the disclosure of which is
incorporated herein by reference. Examples of assay applications
are enzyme-linked immunosorbent assay (ELISA), radioimmunoassay
(RIA) techniques, and lateral flow assays for demonstrating and
assaying an analyte in solution, immunological methods for the
detection of macromolecules in filter blots, immunocytochemical
methods for the study of morphologically intact tissues and cells,
etc. As for the cytochemical applications, they usually target
superficial antigens, as phosphor particles of 0.1-1.0 .mu.m cannot
easily penetrate cell membranes.
[0005] By using phosphors as detectable labels in an assay, several
parameters can be studied and measured at the same time. Not only
is it possible to generate three spectrally separate colours (blue,
green, red) by means of infra red (IR), ultra violet (UV), or
electron excitation, for example, but phosphor emission wavelengths
and intensity aplitudes can be measured, and the number of analytes
to be measured at the same time can become very large
(multiplexing). Time-resolved luminescence assays are comparable in
sensitivity to radioactivity assays. The immunocytochemical use of
phosphor conjugates with capture molecules (analyte-specific
phosphor conjugates) allows a much more sensitive detection of
small quantities of macromolecules in cells. This may be of
importance in both fundamental and diagnostic examination in
various applications.
[0006] Examples of the properties of the phosphors, other than
their high physico-chemical stability, are that they can be
rendered visible by excitation with, for example, IR excitation, UV
light, or with an electron beam, and that the luminescence of the
phosphor-capture molecule conjugates, such as phosphor-antibody
conjugates, does not decrease during excitation (no bleaching). In
addition, the luminescence belongs to the relatively slow
luminescence (phosphorescence). The luminescence of phosphors can
be observed with microscope fluorimeters and flow cytometers. These
can be modified for time-resolved luminescence assays in a
relatively simple manner. The use of phosphor-capture molecule
conjugates makes it possible to assay a plurality of analytes
simultaneously, because the luminescence of phosphors is not only
well separated spectrally (blue, green, red), but also exhibits
measurable differences in decay times.
[0007] Prior multiplexing was generally performed by selective
excitation and/or detecting the emission wavelength of the
different phosphors. Simultaneous detection of multiple phosphors
is possible, at least where the phosphors have the same excitation
bands or different emission bands.
[0008] Currently there is a need for more rapid, ultrasensitive,
and specific assays and methods that can image and detect multiple
analytes in a sample in a single test assay readout. Because prior
phosphor particles were not uniform in their morphology, size,
and/or composition, it was not possible to detect analytes based on
the unique optical lifetime signature of each type of phosphor in
the conjugate being used as detectable label in an assay.
SUMMARY OF THE INVENTION
[0009] Disclosed herein, in certain embodiments, are methods for
detecting one or more analytes in a sample (multiplexing),
comprising the steps of: (a) contacting the sample with two or more
types of phosphor particles, wherein each type of phosphor is
conjugated to a capture molecule specific for an analyte of
interest, to separately label each analyte of interest; (b)
separating phosphor particles bound to the analytes from unbound
phosphor particles; and (c) detecting each labelled analyte by the
unique optical lifetime signature of the corresponding phosphor.
The phosphor particles conjugated to each type of capture molecule
have unique and uniform morphology, size, and/or composition,
producing a unique optical lifetime signature.
[0010] Also disclosed are methods for detecting one or more
analytes in a sample, wherein the detecting step is performed in a
single readout. In another embodiment, the methods further
comprise, before the step of contacting the sample with two or more
types of phosphor particles, the step of capturing the one or more
analytes on an analyte-specific capture molecule attached to a
substrate.
[0011] Also disclosed herein are methods for detecting one or more
analytes in a sample using two or more types of phosphor particles,
wherein the phosphor particles are up-converting phosphor
particles. These are up-converting phosphor particles comprise at
least one rare earth element and a phosphor host material.
[0012] In some embodiments, the disclosed multiplexing methods for
detecting one or more analytes in a sample are cell sorting
methods, and the analytes of interest are cells. In other
embodiments, the methods for detecting one or more analytes in a
sample are used in flow cytometry.
[0013] In certain embodiments, the disclosed multiplexing methods
can be performed using phosphor particles that are about 30 nm to
about 400 nm in size.
[0014] Further disclosed are methods for multiplexed analyte
detection in a sample, wherein the sample comprises a bodily fluid.
In some embodiments, the bodily fluid sample can comprise blood
serum, saliva, tissue fluid, or urine.
[0015] Also disclosed herein are assay kits for detecting one or
more analytes in a sample. The disclosed kits include two or more
types of phosphor particles conjugated to capture molecules
specific for each analyte, and the phosphor particles conjugated to
each type of capture molecule have unique and uniform morphology,
size, and/or composition.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 shows Upconversion lifetime multiplexed detection of
three unique particles (hexagonal, bi-pyramidal, and spherical) in
a single suspension. Lifetimes can be tuned through particles'
morphology, composition, and/or size.
[0017] FIG. 2 depicts Upconversion multiplexed spectral lifetime
detection of various rare earth nanoparticle compositions in a
single readout.
[0018] FIG. 3 shows time-dependent emission from 625 ng/.mu.L
concentration (FIG. 3A) and from 40 pg/.mu.L concentration (FIG.
3B).
[0019] FIG. 4 depicts UCNP titration curve from Series A lateral
flow assay (LFA) strips, after normalizing for additional gain on
the low end (lowest 3 data points).
[0020] FIG. 5 shows the 625 ng/.mu.L UCNP strip, where the focus in
the z-axis was adjusted in increments of 0.025'' and then converted
to mm, and the resulting signal was plotted and fit to a Gaussian
with a width of 2.8407.+-.0.314 mm, showing relatively large
insensitivity to z-axis.
[0021] FIG. 6 depicts the results of measuring and plotting on the
graph the signal in the transverse direction (presumably the
direction of flow in a real assay) at optimal z-axis position in
increements of 0.005''. The signal was fit to a Gaussian, with
width=0.45092.+-.0.0934 mm.
[0022] FIG. 7 shows a lateral flow test strip. FIG. 7A shows the
schematic of the UCP-rT24H lateral flow (LF) strip: Test line (T)
200 ng rT24H and Flow Control line (FC) 100 ng protein-A. FIG. 7B
depicts the LF protocol for antibody detection (referred to as
consecutive flow, CF) with the three sequential flow steps
indicated.
[0023] FIG. 8 depicts optimization of rT24H capture antigen load of
the T-line. Performance of the up-conversion nanocrystals
(UCNC)-rT24H assay with a standard reference panel of cysticercosis
serum samples. The T-line signal (panel A) and Ratio value (panel
B) indicate an optimum for the 2.5 U sample with the 200 ng rT24H
Test. Assay results are presented as normalized assay values,
percentage of the highest signal obtained with the 100 U infection
reference.
[0024] FIG. 9 shows comparison of the UCP-rT24H assay with the
ELISA (single blind evaluation with 141 clinical samples). FIG. 9A
shows comparison of Ratio values obtained with the 40 nm sized YF
UCNC particles with the ELISA OD450 values. FIG. 9B depicts
comparison of Ratio values obtained with the 400 nm sized YOS UCNC
particles with the ELISA OD450 values. FIG. 9C shows Spearman
ranking of the UCNC-rT24H Ratio values obtained with both types of
UCNC particles, and the grey box indicates samples scoring values
below the low specificity threshold (US resident control
group).
[0025] FIG. 10 shows the lower limit detection with sub-micron and
nano-sized UCP particles -400 nm submicron UCP (FIG. 10A) and 40 nm
nano UCP (FIG. 10B).
[0026] FIG. 11 shows the transmission of electron image of
comparative phosphor particles. FIG. 11A shows transmission
electron image of non-uniform-shape 400 nm NaYF.sub.4:Yb.sup.3+,
Er.sup.3+ up-converting phosphor particles (Corstjens et al., IEE
Proc Nanobiotechnol 152: 64-72 (2005)). FIG. 11B shows transmission
electron image of the uniform hexagonal 40 nm NaYF.sub.4:Yb.sup.3+,
Er.sup.3+ up-converting phosphor particles used as the detectable
label for the proof-of-concept study in Example 2.
[0027] FIG. 12 shows an image of the modified FluoroCount Packard
benchtop reader for scanninig multiple LF strips.
[0028] FIG. 13 shows an image of a portable, custom adapted,
lightweight ESEQuant lateral flow strip reader LFR.
DETAILED DESCRIPTION
[0029] The invention relates to new methods and assays for
multiplexed detection of analytes using detectable phosphor labels
where particular phosphor particles are characterized by a uniform
morphology, a uniform size, and/or composition and possesses their
own unique optical spectral lifetime signatures. The detectable
phosphor labels are phosphor-capture molecule conjugates. Any
capture molecule known in the art may be used and applied to the
phosphor using means known in the art. See, for example, U.S. Pat.
No. 5,043,265 (Tanke et al.) and U.S. Pat. No. 5,043,265 (Zarling,
et al.), the disclosures of which are incorporated herein by
reference. For each unique phosphor-capture molecule conjugate used
as a detectable phosphor label in the invention, the particular
phosphor is characterized by its own uniform morphology, a uniform
size, and/or composition and possesses its own unique optical
spectral lifetime signatures. The conjugate molecule is specific
for a particular analyte of interest. This allows for multiple
analytes to be detected by using multiple and different detectable
phosphor labels. The presence or absence of a particular analyte is
determined by whether or not the unique optical lifetime signature
of the particular phosphor is observed.
[0030] The detectable phosphor labels of the invention may be used
in any assay form where phosphor labels or particulate labels are
used, such as those discussed above. Accordingly the methods and
assays of the invention using the detectable phosphor labels can
have a variety of applications, including chemical and biological
detection and imaging. The described methods can be used for
detection and/or imaging of multiple unique chemical or biological
markers simultaneously in a single test assay and single readout.
The assay may be a qualitative or a quantitative assay.
[0031] The detectable phosphor labels of the invention are prepared
by conjugating a capture molecule, such as, but not limited to, an
antibody or a protein specific for the analyte being screened for,
to the phosphor particles as is known in the art. The phosphor
particle may be coated with a surface modifier such as a poly
(acrylic acid) polymer or an inert silica layer to allow or improve
the cojugation of the capture molecule to the particle surface. The
phosphor particles are rare earth nanocrystals or submicron
phosphor monodisperse particles having uniform morphology and a
uniform size. Such phosphor particles can then be detected in
various assay formats and identified by their unique optical
lifetime signature.
[0032] The use of phosphors as labels provides a rapid,
multiplexed, and specific assay platform capable of detecting low
levels of circulating analyte targets in human and non-human
biological samples, such as, but not limited to, blood, saliva,
tissue, and urine.
[0033] One such platform described herein, refers to a lateral flow
assay format where the phopshor particle in the detectable label
may be an up-converting phosphor (UCP) but where, instead of
detecting the up-converting emission wavelength, the phosphor
lifetime signature is detected. Up-conversion luminescence using
rare-earth doped nanocrystals and submicron particles is
increasingly being used in commercial and industrial applications
and more recently has been used in life science applications.
Up-conversion luminescence is based on the absorption of two or
more low-energy (longer wavelength, typically infrared) photons by
a nanocrystal followed by the emission of a single higher-energy
(shorter wavelength) photon. Some aspects of lateral flow assays
using UCP's--but not the use of phosphor labels where the phosphor
lifetime signature is detected--have been described in Corstjens et
al. (2014), Feasibility of Lateral Flow Test for Neurocysticercosis
Using Novel Up-Converting Nanomaterials and a Lightweight Strip
Analyzer, PloS Negl. Trop. Dis. 8(7):e2944.
Doi:10.1371/journal.pntd.0002944, which is incorporated here by
reference.
[0034] The ability to adjust the size, morphology, absorption,
emission, rise time, decay time, power density, and other
properties of phosphor particles such as up-converting nanocrystals
(UCNC) or submicron phosphor particles enables the formation of
materials with an infinite amount of distinctive signatures. The
versatility of the rare earth UCNC platform significantly increases
the ability to have a broad detection capability using a single
reader system. Additionally, the ability to optically tune the rare
earth nanoparticle or submicron particle unique spectral
fingerprints provides limitless multiplexing capabilities. Phosphor
particles such as up-converting crystals and nanocrystals with
sizes ranging from 5 nm to 400 microns have been prepared, and the
morphology of the crystals can be spherical, hexagonal, cubic,
rod-shaped, or diamond-shaped. UCNC do not photobleach and allow
high power density excitation over long term exposure with
simultaneous signal integration. They can be stored indefinitely
without a decrease in light emitting efficiency and thus they allow
repeated irradiation and analysis.
[0035] Suitable rare earth crystalline phosphors are the
morphologically and size uniform, monodiseprse phosphor particles
described in U.S. Pat. No. 9,181,477, which is incorporated herein
in its entirety. These phosphors include up-converting and
down-converting phosphor compositions. Sunstone Nanocrystals from
Intelligent Material Solutions.RTM. Inc. (IMS), for example, are a
proprietary series of rare earth-doped nanocrystals of small size,
high quantum efficiency, and high photoluminescent intensity
functionalized for use in industrial and life sciences
applications. These nanocrystals possess unique and inherent atomic
states that allow the conversion of various wavelengths of light
energy up and down the electromagnetic spectrum. Sunstone
Nanocrystals are synthesized using specific compositions of
individual rare earths and other host elements. Up-conversion
luminescence by Sunstone Nanocrystals occurs through a combination
of a trivalent rare-earth sensitizer (e.g., Yb, Nd, Er, or Sm) as
the element that initially absorbs the electromagnetic radiation
and a second lanthanide activator (e.g., Er, Ho, Pr, Tm) ion in an
optical passive crystal lattice that serves as the emitting
elements. By varying the concentrations and ratios of rare earths,
different emission spectra can be elicited from the same
combination of elements. By varying the concentrations and ratios
of rare earths, different emission spectra can be elicited from the
same combination of elements.
[0036] Multiple nanoparticles and microparticles possessing unique
lifetimes and/or morphologies can be combined and introduced into
or onto an analyte providing a unique detectable label that can be
used for analyte detection. The rare earth nanoparticles are
ideally suited for the proposed application because of their
relatively long phosphorescence lifetime decays attributed to the
trivalent rare earth (or lanthanide) metals.
[0037] Phosphorescence is emission of luminescence which involves
an internal conversion process called intersystem crossing for
populating triplet states from the lowest excited singlet state and
returning to ground state yielding slow emissions decay rates
(microseconds to milliseconds) compared to the fluorescence of
organic fluorophores which emit photons returning to the ground
state from an excited singlet state with rapid lifetime decays in
the nanosecond time scale. "Pulse" or "time-domain" lifetime
measurements can be used as opposed to frequency-domain (or phase
modulation).
[0038] Relatively inexpensive time-gating approaches can be used
instead for measurements of either steady-state luminescence or
intensity decays (lifetimes) on timescales, in the case of
phosphorescence, which conform to the microsecond speeds of most
current image detector arrays such as CMOS and CCD linear or area
sensor arrays as well as photodiode arrays, all of which also offer
the potential benefit of being used to spectrally discriminate
multiple single exponential decays differing according to their
emission wavelengths. In this application, this advantage is
exploited to enable a "multiplexing" capability whereby a mixture
of rare earth nanoparticles differing in emission wavelengths
(under single 980 nm excitation), as well as differing in
pre-determined values for their emission intensities and average
lifetimes are used to develop a capacity for a very large number of
anti-counterfeiting coding sequence combinations.
[0039] Typically, an excited state population decays exponentially
after turning off the excitation pulse by first-order kinetics,
following the decay law, I(t)=I.sub.0 exp (-t/.tau.) whereby for a
single exponential decay I(t)=time dependent intensity, I.sub.0=the
intensity at time 0 (or amplitude), and .tau.=the average time a
nanoparticle remains in the excited state (or <t>) and is
equal to the lifetime. (The lifetime, .tau. .sigma. the inverse of
the total decay rate, .tau.=(T+k.sub.nr).sup.-1, where at time t
following excitation, T is the emissive rate and k.sub.nr is the
non-radiative decay rate). In general, the inverse of the lifetime
is the sum of the rates which depopulate the excited state. The
luminescence lifetime can be simply determined from the slope of a
plot of InI(t) versus t (equal to 1/.tau.) can also be the time
needed for the intensity to decrease to 1/e of its original value
(time 0). Thus, for any given known emission wavelength, a number
of parameters fitting the exponential decay law can be monitored
for their use in developing anti-counterfeiting codes.
[0040] The lifetimes of the phosphor particles can be precisely
tuned for each emitted wavelength of the particle determined by the
particle morphology and composition. For instance, particles of
identical compositions have been shown to exhibit unique lifetime
responses to an excitation source as can be seen in FIG. 1. Samples
A (spherical, 220 .mu.s) and E (hexagonal, 710 .mu.s) possess
identical compositions of NaYF.sub.4:Yb,Tm, but have different
morphologies yielding a unique lifetime signature.
[0041] As mentioned above, it is desirable to combine known
emission wavelengths, intensity amplitudes, and lifetimes in this
regard. FIG. 2 provides an example of the multiplexing capabilities
of the nanoparticles. The figure depicts multiple nanoparticle
emission signatures and lifetime decay profiles detected in a
single sample or substrate for detectable labels of the invention
which may be used in a multiplexed assay.
[0042] Disclosed herein, in certain embodiments, are methods for
detecting one or more analytes in a sample (multiplexing). The
disclosed methods include the steps of: (a) contacting the sample
with two or more types of phosphor particles, wherein each type of
phosphor is conjugated to a capture molecule specific for an
analyte of interest, to separately bind and label each analyte of
interest; (b) separating phosphor particles bound to the analytes
from unbound phosphor particles; and (c) detecting each labelled
analyte by the unique optical lifetime signature of the
corresponding phosphor. In the disclosed multiplexing methods, the
phosphor particles conjugated to each type of capture molecule have
unique and uniform morphology, size, and/or composition, producing
a unique optical lifetime signature.
[0043] Also disclosed are methods for detecting one or more
analytes in a sample, wherein the detecting step is performed in a
single readout. In some embodiments, the methods further comprise,
before the step of contacting the sample with two or more types of
phosphor particles, the step of capturing the one or more analytes
on an analyte-specific capture molecule attached to a
substrate.
[0044] Also disclosed herein are methods for detecting one or more
analytes in a sample using two or more types of phosphor particles,
where the phosphor particles are up-converting phosphor particles.
These up-converting phosphor particles can comprise at least one
rare earth element and a phosphor host material.
[0045] In some embodiments, the disclosed multiplexing methods for
detecting one or more analytes in a sample are cell sorting
methods, and the analytes of interest are cells. In certain other
embodiments, the methods for detecting one or more analytes in a
sample are used in flow cytometry.
[0046] In certain embodiments, the disclosed multiplexing methods
can be performed using phosphor particles that are about 30 nm to
about 400 nm in size.
[0047] Further disclosed are methods for multiplexed analyte
detection in a sample, wherein the sample comprises a bodily fluid.
In some embodiments, the bodily fluid sample can comprise blood
serum, saliva, tissue fluid, or urine.
[0048] Also disclosed herein are assay kits for detecting one or
more analytes in a sample. The disclosed kits include two or more
types of phosphor particles conjugated to capture molecules
specific for each analyte, and the phosphor particles conjugated to
each type of capture molecule have unique and uniform morphology,
size, and/or composition.
EXAMPLES
[0049] The following assays, methods, as well as ingredients,
processes, and procedures for practicing the assays and methods
disclosed herein correspond to that described above. The procedures
below describe with particularity illustrative, non-limiting
embodiments of the disclosed assays and methods.
Example 1
[0050] Phosphor lifetime detection was performed using 35 nm
NaYF.sub.4:YbEr nanocrystals sprayed onto a nitrocellulose membrane
in a standard dilution series. FIG. 3A shows time-dependent
emission from 625 mg/.mu.l concentration, and FIG. 3B depicts
time-dependent emission from 40 mg/.mu.l concentration.
[0051] The photomultiplier tube (PMT) gain was initially 0.4V
control voltage (5.times.103), but was adjusted upward for the low
range to 0.9V control voltage (1.5.times.106). The electronic gain
was 105. No light-tight enclosure was used, so low level ambient
light was present. The excitation source was a 2 W 980 nm laser,
and the emission was filtered with a 700 nm short pass filter. The
laser was turned on and off to allow for gated lifetime
detection.
[0052] The time-dependent emission from the 625 ng/uL strip (FIG.
3A) was fit to a double-exponential, and the following parameters
were found to best fit the data:
[0053] A1=0.53449.+-.0.0921
[0054] tau1=0.15816.+-.0.0108 ms
[0055] A2=0.80529.+-.0.0838
[0056] tau2=0.39765.+-.0.0334 ms
[0057] The peak amplitude as a function of UCNP concentration, is
shown in FIG. 4, which shows UCNP titration curve from Series A LFA
strips, after normalizing for additional gain on the low end
(lowest 3 data points).
[0058] To examine the sensitivity to transverse and z-axis focus,
the signal from the 625 ng/uL spot was measured as a function of
distance along the z-axis (FIG. 5) and transverse (FIG. 6)
direction.
[0059] FIG. 5 shows the 625 ng/uL UCNP strip. The focus in the
z-axis was adjusted in increments of 0.025'' and then converted to
mm. The resulting signal was plotted and fit to a Gaussian with a
width of 2.8407.+-.0.314 mm, showing relatively large insensitivity
to z-axis.
[0060] The signal in the transverse direction (presumably the
direction of flow in a real assay) was measured at optimal z-axis
position in increements of 0.005'' and plotted (FIG. 6). The signal
was fit to a Gaussian, with width=0.45092.+-.0.0934 mm.
Example 2
[0061] The following example has demonstrated proof-of-concept
development with respect to usability and sensitivity of 40 nm
sized NaYF.sub.4:Yb.sup.3+, Er.sup.3+ polyacrylic acid-coated
up-converting phosphor particles as UCNC reporter materials from
Intelligent Material Solutions and analyzer equipment, where an
up-conversion lateral flow assay format was used to detect
neurcysticercosis. This proof-of-concept involved a single reporter
material using a lateral flow assay format and detected the
up-converted visible emission from the UCNC reporter materials.
FIG. 11 shows the transmission of electron image of comparative
phosphor particles. FIG. 11A shows transmission electron image of
non-uniform-shape 400 nm NaYF.sub.4:Yb.sup.3+, Er.sup.3+
up-converting phosphor particles (Corstjens et al., IEE Proc
Nanobiotechnol 152: 64-72 (2005)). FIG. 11B shows transmission
electron image of the uniform hexagonal 40 nm NaYF.sub.4:Yb.sup.3+,
Er.sup.3+ up-converting phosphor particles used as the detectable
label for this proof-of-concept study. FIG. 12 shows an image of
the modified FluoroCount Packard benchtop reader for scanninig
multiple LF strips. FIG. 13 shows the lateral flow strip reader and
the NaYF.sub.4:Yb.sup.3+, Er.sup.3+ up-converting phosphor
particles.
[0062] Neurocysticercosis is a frequent parasitic infection of the
human brain, occurring in most of the world, which requires imaging
of the brain to diagnose. It is also the most frequent preventable
cause of epilepsy in developing countries. To determine the burden
of the disease and to simplify diagnosis, a low-cost, highly
sensitive detection platform has been developed. The availability
of a rapid serological diagnosis that targets stage-specific
antibodies for human cysticercosis is considered very helpful in
control programs for estimating the burden (sero-prevalence) of
disease in susceptible population groups. A low-cost rapid
diagnostic test could also be applied to determine sero-prevalence
rates in pigs to assess interruption of transmission.
[0063] The up-converting phosphor lateral flow (UCP-LF) assay
format used in this study was a consecutive flow assay (the
sequential flow of sample, buffer, and UCP reporter particle), such
as described in U.S. Pat. No. 7,858,396, which is incorporated
herein by reference. It included the use of a generic UCP reporter
(e.g., protein-A coated UCP particles) to detect human antibodies
on a single lateral flow strip. The general protocol applied for
the UCNC-LF assay used for the majority of the experiments in this
study implied a dilution of sera in assay buffer such that 14
undiluted serum was delivered to the LF strips during the first
flow step of the CF protocol (FIG. 7).
[0064] Serum Sample Load:
[0065] The performance of the UCNC-rT24H assay was first assessed
with a set of sera with different reactivity ranging from 1 to 100
Units as determined by ELISA. The 2.5 Units sample is indicative
for the targeted lower limit of detection (LLOD), a sample with low
antibody titer. The general protocol applied for the UCNC-LF assay
used for the majority of the experiments in this study, implied a
dilution of sera in assay buffer such that 1 .mu.L undiluted serum
was delivered to the LF strips during the first flow step of the CF
protocol (FIG. 7). The assay appeared to perform similar to
previously described UCNC-CF antibody assays indicating high degree
of flexibility towards sample input.
[0066] Amount of rT24H Antigen on the Test (T-)Line:
[0067] Often, the major production cost of LF-based assays is
associated with the capture antigen on the T-line. The density
(amount) of specific-antigen must be enough to prevent passing of
the target molecules without interacting with the capture antigen.
However, excess antigen on the T-line will lead to poor
immobilization of the capture antigen on the LF strip, which can
result in the unexpected loss of signal. For the UCNC-rT24H assay
the T-line is comprised of purified rT24H. FIG. 8A shows the result
of a typical experiment, indicating lower T-signal due to release
when using 400 ng of rT24HNS antigen (per 4 mm width).
[0068] All assays were performed with the same amount of UCNC
label, and T-signal values were normalized to the highest T-signal
measured with the 100 Units sample; achieved with the LF strips
containing an rT24H density of 100 ng, the 200 ng strips scored
only a slightly lower signals. Differences become more pronounced
when looking at normalized Ratio values (T-line signal divided by
FC-line signal). An optimum around the targeted lower limit of
detection (LLOD) of 2.5 Units with LF strips containing a T-line
comprised of 200 ng rT24H seems apparent. A large difference
between the zero and the sample indicative for the LLOD is
essential to determine a solid assay cutoff threshold. The relative
differences in Ratio values determined for the 0 and 2.5 Units
samples were a factor of 2.16, 5.20 and 3.32 for the 100, 200 and
400 ng strips, respectively; corresponding A450 ELISA values (not
shown) indicated a factor of 2.86. These values may differ when
using differently sized UCNC particles; the experiment shown in
FIG. 8 was performed with 400 nm particles, similar results were
observed with the 40 nm particles. An additional constraint to
consider is the sensitivity of the applied UCNC-LF strip scanner,
which is the lowest UCNC signal that can be measured with the
current UCNC reader.
[0069] Cutoff Threshold and Clinical Parameters:
[0070] The established UCNC-LF assay conditions used to validate
the UCNC-rT24HNS neurocysticercosis antibody assay involved the use
of 4 mm width LF strips with a T-line of 200 ng rT24H and the
addition of the equivalent of 1 uL undiluted serum sample and 500
ng UCNC protein-A coated reporter particles. Testing of the
clinical samples was performed in parallel with using both types of
UCNC reporter particles: The 40 nm NaYF.sub.4:Yb,Er particles with
poly(acrylic acid) surface and the 400 nm sized
Y.sub.2O.sub.2S:Yb,Tm particles with a silica coated
carboxyl-functionalized surface.
[0071] Cutoff threshold: In order to assess clinical specificity,
the assay cutoff threshold was evaluated with two sets of sera
samples from healthy individuals following a protocol as described
earlier (Corstjens et al., J. Clin. Microbiol. 2008) implying the
definition of a low and high specificity cutoff threshold. The
UCNC-rT24H ratio values were determined for both sample sets (92
Dutch blood donors and 78 healthy US residents) using both types of
UCNC particles. Table 1 summarizes the determined values; the low
specificity cutoff threshold was defined as the average Ratio value
plus two times the standard deviation and the high specificity
cutoff threshold was defined as the highest Ratio value in the
control group plus two times the standard deviation.
TABLE-US-00001 TABLE 1 Cutoff threshold of the UCNC-rT24H assay. 92
Dutch blood donors 78 USA residents 40 nm YF 400 nm YOS 40 nm YF
400 nm YOS Ratio: Average Value (AV) 0.029 0.008 0.046 0.009 Ratio:
Highest Value (HV) 0.110 0.026 0.186 0.033 Standard Deviation: (SD)
0.021 0.005 0.032 0.007 Low specificity cutoff: AV + 2SD 0.070
0.019 0.109 0.023 High specificity cutoff: HV + 2SD 0.151 0.037
0.231 0.047
[0072] Samples generating Ratio values below the low specificity
cutoff were considered antibody negative with the UCNC-rTH24 test,
samples above the high specificity cutoff were considered antibody
positive. To determine the most likely classification of samples
generating signals in between the low and high specificity cutoff,
the determined threshold values need to be evaluated with a large,
statistically relevant, set of confirmed positives. The significant
difference in cutoff values when using YF or YOS UCNC particles is
a technical issue that can be regulated by changing assay
conditions (e.g., the amount of UCNC particles or the amount of
rT24H on the Test line). The observed smaller difference in cutoff
value between the two sets of healthy individuals tested with the
same UCNC particles may indicate an effect based on cultural
behavior and/or ethnicity.
[0073] Single Blinded Assay Validation:
[0074] Validation of the assay was accomplished with the selection
banked serum samples of 63 classified cases of neurocysticercosis
randomly arranged between the set of 78 serum samples from healthy
US resident. The resulting 141 samples were tested with both types
of UCNC particles in a single blind experiment. Obtained UCNC-rT24H
Ratio values were plotted against the corresponding rT24H ELISA
OD450 values (FIG. 9) showing a good correlation between the UCNC
and ELISA.
[0075] The test conditions for the ELISA were set for best
resolution in the low reactive range, implying a max OD450 value of
4 and thus no discrimination between high reactive samples. As
observed when testing the cutoff threshold samples, the Ratio
values determined with the 40 nm YF UCNC particles on average
differed by a factor of .about.4 compared to the Ratio values
determined with the 400 nm YOS UCNC particles. Qualitatively both
type of UCNC particles seem to perform quite similar, whereas
quantitatively the 40 nm YF UCNC particles seem to correlate
somewhat better with the ELISA. FIG. 9C, which is a scatter plot of
the results obtained with the two types of UCNC particles, shows
the Spearman correlation (R2 is 0.90) of the Ratio values by rank
order. The relatively large scattering of points in the lower range
is directly linked to the low Ratio values measured for the
non-reactive samples (indicated by the grey box).
[0076] Clinical Sensitivity and Specificity:
[0077] Application of the low and high specificity thresholds
(Table 2) as determined with the 170 healthy controls indicated a
number of UCNC-rT24H false positives and false negatives.
[0078] A sample was classified false positive (FP) when it is part
of the set of healthy controls with a Ratio value score above the
cutoff threshold, a sample was classified false negative (FN) when
it is part of the set of defined cysticercosis set (with 2 or more
cysts identified by microscopy). When using both low and high
specificity thresholds, an indecisive (IND) or potentially positive
group can be identified from samples scoring Ratio values between
the low and high specificity threshold (Table 2).
TABLE-US-00002 TABLE 2 Performance of the UCNC-rT24H assay. UCNC
Reporter 40 nm YF UCNC particles 400 nm YOS UCNC particles Control
group Dutch donors US residents Dutch donors US residents
Specificity threshold Low High Low High Low High Low High
Indecisive (IND) 0 8 0 10 0 7 0 8 False negative (FN), IND included
1 1 3 1 2 4 4 0 FN without IND 1 9 3 11 2 11 4 8 False positive
(FP) in Dutch group 6 0 1 0 5 0 2 0 FP in US group 18 1 3 0 7 0 2 0
FP, Dutch + US group 24 1 4 0 12 0 4 0 Clinical Sensitivity
(Sn).sup.a 98.4% 87.5% 95.5% 85.1% 96.9% 85.1% 94.0% 88.7% Clinical
Specificity (Sp).sup.b 87.6% 99.4% 97.7% 100.0% 93.4% 100.0% 97.7%
100.0% Youden's index, J 0.863 0.870 0.933 0.851 0.905 0.851 0.919
0.887 .sup.aSn was calculated dividing the number of true positives
(TP) by the sum of the number of TP + FN; TP are the 63 classified
cystycercosis samples (Peru sample set) .sup.bSp was calculated
dividing the number of true negatives (TN) by the sum of the number
of TN + FN; TN are the 170 healthy control samples (Dutch and US
sample set)
[0079] By definition, at 100% clinical specificity (Sp) all 170
healthy control samples should score a Ratio value below the cutoff
threshold. For the assay with e.g. the 40 nm YF UCNC particles,
this is achieved when using the high specificity threshold
determined for the US resident group. Clinical sensitivity (Sn) of
the assay then drops to 85%; for the 400 nm YOS UCNC particles at
100% specificity the sensitivity is 89%. The highest sensitivity,
obtained when applying the low specificity threshold determined for
the Dutch blood bank donors, is 98% with a specificity of 88%. The
actual required cutoff threshold is depending of the clinical
sensitivity and specificity required demanded for this assay; in
this respect the area of receiver operating characteristic (ROC)
curves of the UCNC-rT24H indicate an area of 0.99 for both types of
UCNC particles. In this particular test the low specificity
threshold determined for the US resident group seems to deliver
acceptable sensitivity/specificity levels of 96%/98% (Youden's
index, J=0.933) and 94%/98% (J=0.919) for the 40 and 400 nm UCNC
particles, respectively. For both particles, these numbers imply a
positive and negative predictive value of 94% and 98%,
respectively. With the ELISA 96% sensitivity is achieved with 94%
specificity (J=0.898). These numbers indicate at least equivalent
performance of the UCNC-rT24H assay as compared to the ELISA.
Moreover, results show that in consecutive flow based assays [41]
the 40 nm YF UCNC show significantly improved sensitivity over the
400 nm YOS UCNC particles.
[0080] Submicron-Versus Nano-Sized UCNC Particles:
[0081] The potential of a new type of UCNC particles, nano-sized 40
nm particles, was tested on LF strips containing T-lines with 200
ng rT24H antigen. In these experiments the 1 Unit standard sample
was included rather than the 2.5 Units standard to allow
exploration below the targeted LLOD, set a 2.5 Units. Four standard
samples (0, 1, 10 and 100 Units) were diluted 10- and 100-fold in
NHS and analyzed with UCNC-T24HNS assay using UCNC conjugates made
with the 40 nm and 400 nm reporter particles. FIG. 10 shows the
result of an experiment performed in triplicate, assay values were
normalized to the highest Ratio value obtained with the 100 Units
sample. Overall, the applied test conditions (500 ng UCNC conjugate
per strip) seem to be in favor of the 40 nm nano-particles. This is
demonstrated by the increase of the signal strength observed for 1
to 10 Units for the 40 nm particles: a factor of 7.69 versus 3.75
increase for the 40 and 400 nm particles, respectively. The 500 ng
UCNC particles per LF strip matched well with the lightweight
UCNC-Quant strip reader available for the analysis. IMS plans on
further developing its nanocrystal synthesis and nanocrystal size
and composition to increase the overall quantum efficiency (QE) of
the UCNCs. Adjusting the nanocrystal composition as well as
utilizing core shell structures, it is expected to see 10.times.
increase in brightness.
* * * * *